Lignocellulosic feedstocks are a promising alternative to corn starch for the production of fuel ethanol. These raw materials are widely available, inexpensive and several studies have concluded that cellulosic ethanol generates close to zero greenhouse gas emissions.
However, these feedstocks are not easily broken down into their composite sugar molecules. Recalcitrance of lignocellulose can be partially overcome by physical and/or chemical pretreatment. An example of a chemical pretreatment is steam explosion in the presence of dilute sulfuric acid (U.S. Pat. No. 4,461,648). This process removes most of the hemicellulose, but there is little conversion of the cellulose to glucose. The pretreated material may then be hydrolyzed by cellulase enzymes.
The term cellulase broadly refers to enzymes that catalyze the hydrolysis of the beta-1,4-glucosidic bonds joining individual glucose units in the cellulose polymer. The catalytic mechanism involves the synergistic actions of endoglucanases (E.C. 3.2.1.4), cellobiohydrolases (E.C. 3.2.1.91) and beta-glucosidases (E.C. 3.2.1.21) (Henrissat et al, 1994; Knowles et al., 1987; Lynd et al., 2002; Teeri, 1997; Wood and Garcia-Campayo, 1990; Zhang and Lynd, 2004). Endoglucanases hydrolyze accessible glucosidic bonds in the middle of the cellulose chain, while cellobiohydrolases processively release cellobiose from these chain ends. Beta-glucosidases hydrolyze cellobiose to glucose thus minimizing product inhibition of the cellobiohydrolases and endoglucanases.
Beta-glucosidases are produced by many organisms occurring in all five living kingdoms. Generally these enzymes hydrolyze aryl-beta-glucosides, among which is included cellobiose (gluco-beta-(1,4)-glucoside). Some also catalyze transglycosylation reactions to varying extents.
Filamentous fungi, including Trichoderma ssp., Aspergillus ssp., Hypocrea ssp., Humicola ssp., Neurospora ssp., Orpinomyces ssp., Gibberella ssp., Emericella ssp., Chaetomium ssp., Fusarium ssp., Penicillium ssp., Magnaporthe ssp., Chrysosporium ssp., Myceliophthora ssp., Theilavia ssp., Sporotrichum ssp. and Phanerochaete ssp. are effective producers of cellulase enzymes. Many of these organisms secrete beta-glucosidase enzymes. Trichoderma spp. (Trichoderma longibrachiatum or Trichoderma reesei) secrete small amounts of beta-glucosidase I or Cel3A (Chirico et al., 1987) and likely also secrete two other beta-glucosidases, Cel3B and Cel3E (Foreman et al., 2003).
The enzymatic hydrolysis of pretreated lignocellulosic feedstocks is an inefficient step in the production of cellulosic ethanol and its cost constitutes one of the major barriers to commercial viability. Improving enzymatic activity has been widely regarded as an opportunity for significant cost savings.
Cellobiohydrolases are strongly inhibited by cellobiose and to a lesser degree by glucose. Conversion of cellobiose to glucose is a rate-limiting step in cellulose hydrolysis because filamentous fungi, such as Trichoderma reesei, produce very low levels of beta-glucosidase and beta-glucosidases are highly sensitive to glucose inhibition (Chirico et al., 1987; Berghem et al., 1974). One technique for reducing cellulase inhibition is to increase the amount of beta-glucosidase in the system (U.S. Pat. No. 6,015,703), as cellobiose is more inhibitory to cellulases than glucose (Holtzapple et al., 1990; Teleman et al., 1995). However, over-expressing a beta-glucosidase in an organism such as Trichoderma may reduce the production of other cellulase enzymes and, in turn, may limit the rate of cellulose conversion to cellobiose. In addition, this approach does not specifically address the effect of glucose inhibition on beta-glucosidase activity. A complementary approach would be to use a beta-glucosidase with a higher specific activity which is also less sensitive to glucose inhibition. This enzyme would mitigate cellobiose product inhibition, but do so with lower amounts of beta-glucosidase (relative to the amount of cellulase(s)) and maintain its catalytic efficiency in the presence of high glucose concentrations.
Beta-glucosidases from most fungi have binding constants for cellobiose (KG2) that range from 0.2-2.0 mM (Chirico et al., 1987; Berghem et al., 1974; Enari et al., 1981; Christakopoulos et al., 1994). These enzymes are highly sensitive to glucose inhibition; KG values for glucose ranging from 0.6-8.0 mM have been reported for these enzymes. Several microbial beta-glucosidases with higher tolerance to glucose inhibition (KG>8.0 mM) have been reported (Riou et al., 1998; U.S. Pat. No. 6,087,131; Saha et al., 1996; U.S. Pat. No. 5,747,320; Gueguen et al., 1995; Li et al., 1991; Perez-Pons et al., 1994; Chen et al., 1994; U.S. Pat. No. 6,184,018 B1). However, these enzymes generally have a lower affinity for cellobiose (i.e., higher KG2 values). As a result, the concentration of cellobiose at steady state would be higher using these beta-glucosidases, increasing the degree of cellobiose inhibition on cellulase activity. Therefore, these particular glucose tolerant beta-glucosidase enzymes have limited utility for the production of cellulosic ethanol.
In spite of much research effort, there remains a need for improved beta-glucosidase enzymes in order to generate enzyme mixtures with higher sustained hydrolysis activity on pretreated lignocellulosic feedstock. The absence of such improved beta-glucosidase enzymes represents a large hurdle in the commercialization of cellulose conversion to glucose and other soluble fermentable sugars for the production of ethanol and other products.